Research On The Function Of BMSCs Effecting Apoptosis Of Ovarian Granulose Cells

Objective:To study the function of BMSCs on apoptosis of ovarian granulose cells in rats.Methods:BMSCs were isolated from rats.Later,the BMSCs were cultured,purified and identified.60 IU serum gonadotrophin was injected subcutaneously into 3-week-old female SD rats.After 48 h,the rats were killed and the ovaries were removed.Granulose cells were isolated from the ovaries by needle puncture under optical microscopy. Granulose cells were cultured,purified and identified.50% inhibiting concentration(IC50) of cisplatin and time were gotten by MTT. There were three groups in vitro experiment:control group(GO),co-cultured group(GM5) and cisplatin group(G5).5 mg/1 cisplatin was added to granulose cells in group GM5 and G5.BMSCs and granulose cells shared the same medium but remained separated by millicell in group GM5.3 days later, granulose cells were collected for apoptosis analysis and RT-PCR. And,there were four groups in vivo experiment:control group,BMSCs group,estrogen group and youth group.The BMSCs group rats were injected 1～2×106 BMSCs,the control group were injected 2 ml saline,the estrogen group were fed estradiol according to 0.158 mg/(kg.d) last 8 weeks when these rats were perimenopausal period.The youth rats,about 3 month old, were injected 2 ml saline the same to the control ones.After 1 month and 3 month,some rats of the four group were killed.And the left ovary of them were used for TUNEL assay. The rat of apoptotic granulose cells were calculated by microscope.Results:In vitro,the apoptosis rates of group G0,G5 and GM5 were 0.59%,13.04%,4.84%.The expression of survivn was G0>GM5>G5, P21 was G5>G0>GM5,Cmy-c was G0>GM5>G5,Bax was G5>G0>GM5,Bcl-2 was G0>GM5>G5. (An unpaired Student's t-test was used for comparisons the expression of all gene between group G5 and group GM5.The P-value of Bcl-2 and Survivn were more than 0.05,but the P-value of the other genes were less than 0.05.) In vivo, the apoptosis rates of granulose cells in control group,MSC group,estrogen group and youth group were 9.90±3.23%,6.10±2.63%(P=0.075),6.60±3.13%(P=0.139),1.53±1.44%(P=0.001) afer one month.Three month later,the apoptosis rates of the four groups were 4.60±3.51%,8.29±3.35%(P=0.02),10.53±2.30%(P=0.062) and 3.56±1.44%(P<0.001). (An unpaired Student's t-test was used for comparisons between control group and the other three groups.A P-value less than 0.05 was considered to denote significant difference.)Conclusion:BMSCs can protect the granulose cells of perimenopausal rats in vivo and cisplatin-induced granulose cells in vitro. In general,BMSCs can reduce apoptosis of granulose cells.